Black phosphorus boosts wet-tissue adhesion of composite patches by enhancing water absorption and mechanical properties

Wet-tissue adhesives have long been attractive materials for realizing complicated biomedical functions. However, the hydration film on wet tissues can generate a boundary, forming hydrogen bonds with the adhesives that weaken adhesive strength. Introducing black phosphorus (BP) is believed to enhance the water absorption capacity of tape-type adhesives and effectively eliminate hydration layers between the tissue and adhesive. This study reports a composite patch integrated with BP nanosheets (CPB) for wet-tissue adhesion. The patch’s improved water absorption and mechanical properties ensure its immediate and robust adhesion to wet tissues. Various bioapplications of CPB are demonstrated, such as rapid hemostasis (within ~1-2 seconds), monitoring of physical-activity and prevention of tumour-recurrence, all validated via in vivo studies. Given the good practicability, histocompatibility and biodegradability of CPB, the proposed patches hold significant promise for a wide range of biomedical applications.

The 1 H NMR spectra of HAMA and PAA-DA were analyzed to investigate the degree of methacrylation of HA and the grafting ratio of DA onto PAA, respectively.
The sample was dissolved in D2O in an NMR tube, and 1 H NMR measurement was carried out using an NMR spectrometer (Bruker Ascend 400M, Bruker, China).BP nanosheets were observed by SEM (ZEISS SUPRA® 55, Carl Zeiss, Germany) at an accelerating voltage of 10 kV and the transmission electron microscopy (TEM, JEM-3200FS, Beijing, China) at an acceleration voltage of 200 kV.
The graft-ratio of MA on pure HA calculated from 1 H NMR spectra was ~20% (Supplementary Fig. 2a).PAA-DA was prepared in advance, where the graft-ratio of DA was ~68% (Supplementary Fig. 2b).The SEM and TEM images of BP nanosheets showed that BP had a sheet size of ~100 nm -1 μm (Supplementary Fig. 2c and 2d).Supplementary Fig. Subsequently, the mixture was placed under the UV light, resulting in the self-crosslink of HAMA.The hydroxyl groups of PVA could formed hydrogen bonds (HBs) with the functional groups such as NH-groups of HAMA and Gel to further enhance mechanical performance.The initial CPB product was obtained after the sample being dried overnight at room temperature (RT).The PAA-DA was dropwise added on the surface of the dried film to form the topological entanglement.Finally, CPB with triple crosslink network was prepared.

Characterizations of CP and CPB
The morphological characterization of the lyophilized CP and CPB was conducted by the scanning electron microscope with Energy Dispersive Spectrometer (SEM-EDS) (ZEISS SUPRA® 55, Carl Zeiss, Germany) at an accelerating voltage of 5 kV.CPB was directly adhered to harvest nude mice skin to evaluate its contact and flexibility.

Mechanical properties
The tensile tests were carried out by using an Instron tester (Instron Electropuls E10000, USA) at a testing rate of 1 mm min -1 at RT. Young's Modulus, stress strength and elongation at break were derived from the stress-strain curves for comparison (Supplementary Fig. 7).Most tissue adhesives have a low strength at dry state, leading to further decreased mechanical and adhesive performance at wet state.The strength of these patches was highly enhanced due to the triple network compared with a great many adhesive hydrogels 2,3,4 .In addition, the mechanical properties of CPB with various ratios of components were investigated.With higher ratios of PVA or higher graft-rations of HAMA, the Young's modulus and tensile strength were improved while the elongation at break was decreased.CPB with higher ratios of BP nanosheets had improved Young's modulus, tensile strength and elongation at break.Therefore, CPB had enough stiffness and flexibility to maintain the position and ensure satisfactory deformation during tissue movement in a wet condition.

Degradation in vitro
The degradation of CP and CPB was evaluated by placing the patches in a phosphatebuffered saline (PBS) solution (pH = 7.4) with a ratio of 0.1g mL -1 according on ISO 10993-12, followed by putting in a thermostatic water bath bed (70 rpm, 37 °C).The

In vitro cell studies
Cell behaviors were investigated for evaluating the biological influences of various samples with or without NIR light.Sterilized CP and CPB were immersed in a minimum essential medium α (α-MEM, Gibco, USA) supplemented with 10% (v v -1 ) fetal bovine serum (FBS, Gibco, USA) and 1% (v v -1 ) penicillin/streptomycin (Gibco, USA) for 24 h at 37℃ in 5% CO2 atmosphere for extraction according to ISO 10993-12.L929 cells (CCL-1, ATCC, Manassas, VA, USA) were cultured using the extract liquid (diluted 50 times) with the samples in a 96-well culture plate with a density of 5×10 3 cells well -1 in an incubator under 37 °C and 5% CO2.The Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay was conducted after being incubated for 1, 3, 5 and 7 days to determine the cells proliferation according to the manufacturer's instructions and literature 6,7 .In addition, MCF-7 cells (HTB-22, ATCC, Manassas, VA, USA) were cultured using the extract liquid (diluted 50 times) in a 24-well culture plate with a density of 2×10 4 cells well -1 in an incubator under 37 °C and 5% CO2 for 24 h.Then CP and CPB were respectively put at the center of the wells followed by being irradiated by NIR light (808 nm, P=1 W cm -2 ) for 5 minutes.After 6 h incubation, Live/Dead staining assay was carried out to determine the effects of CP and CPB on MCF-7 cells.
Moreover, the Annexin V-FITC/ propidium iodide (PI) double-staining assay was also used to investigate cell death according to the protocol and literature 8 .The samples were detected using flow cytometry (BD FACSCanto II, USA).
At first, the Live/Dead staining results suggested the cells were normal in each group without NIR irradiation (Supplementary Fig. 14a).When being placed under NIR light for 5 minutes (808 nm, P=1 W cm -2 ), the control group and CP group had no obvious change due to no photothermal (PT) effect.On the contrary, most tumour cells in the CPB group were killed due to the PT effect.In addition, the Annexin V-FITC/PI staining results proved that the majority (>95%) of the MCF-7 cell death on the CPB with NIR irradiation group (Supplementary Fig. 14b).These results suggested the potential of CPB for in situ photothermal treatment (PTT).In addition, NIR light was also proved to be biosafe for normal cells.To determine the cytotoxicity of the patches, L929 cells were used to investigate the cell viability and proliferation.The control

Potential application of hemostasis and activity monitor
CP and the commercial products (e.g.Gelatin Sponge and Gauze) were used as control groups for evaluating the hemostatic effect in a normal SD rat liver perforation wound model.A total of 15 SD rats (male, weight of 250-300 g, 7-8 weeks) were randomly divided into the CP group, Gelatin Sponge group, and Gauze group.Then the livers of the rats were lifted and placed on the surface of preweighted filter paper, and a circular perforation wound (diameter of 6 mm) was created for hemorrhage.The sample (diameter of ~10 mm) was weighted in advance.Next the corresponding sample in each control group was directly adhered to the bleeding site and the hemostatic process was recorded with a digital camera.The blood loss was calculated by determining the total weight of the blood absorbed by the filter paper and the sample, respectively.Similarly, the hemostatic effect of CP, the commercial Gelatin Sponge and Gauze were evaluated by a normal SD rat heart perforation wound model, where the hearts of rats were lifted and a circular perforation wound (diameter of 6 mm) was created for hemorrhage.The corresponding sample (diameter of ~10 mm) in each control group was immediately adhered to the bleeding sites, and the state was recorded with a digital camera.

2 1 H
NMR (400 MHz, D2O) spectra of (a) MA-grafted HA and (b) DA-grafted PAA.(c) SEM and (d) TEM images of the BP nanosheets.The images were repeated at least twice with consistent results.Preparation of the CPB Supplementary Fig. 3 Schematic illustration of preparation of CPB.HAMA, PVA, BP, EDC, NHS and I2959 were mixed first.Gel solution was added into the mixture with fast stirring followed by being poured into a glass mold.The inter-crosslink bonds between HAMA and Gel formed due to the presence of the EDC/NHS linker.

Fourier
transform infrared spectrophotometer (FTIR) and Raman spectra were performed to confirm the introduction of BP nanosheets by a FTIR spectrometer (Thermo Nicolet iS5, USA) and a Raman Microscope equipment (Thermo Fisher DXR2 xi, USA) with 532 nm laser excitation at RT, respectively.XPS was performed to analyze the protonated and deprotonated forms of the amine-containing and phosphorus-containing chemicals in CPB by a XPS spectrometer (Thermo Scientific Nexsa, USA), using an Al Kα (λ = 0.83 nm, hυ = 1486.6eV) X-ray source operated at 72 W. Supplementary Fig. 4 SEM and EDS images of CP and CPB with various contents of BP nanosheets: CP: 0 mg, CPB-0.2:0.2 mg, CPB-0.6:0.6 mg, CPB: 1 mg, CPB-1.2:1.2 mg.Red: C; Green: O; Blue: P. The micrographs were repeated at least twice with consistent results.Supplementary Fig. 5 Characterizations of CP and CPB.(a) Flexibility.(b) FTIR.Black arrow from left to right: the characteristic bands at 1086, 1024 and 947 cm -1 , respectively.(c) Raman spectra.CPB: A 1 g at ∼360.4 cm −1 , B 2 g at ~431.8 cm −1 , A 2 g at ~460.7 cm −1 .(d) The nitrogen binding energies (N1s) in the XPS spectra of CP.
specimens were taken out and dried in a vacuum oven at RT for 48 h every week before weighting the samples.Supplementary Fig. 9 Degradation in vitro of CP and CPB in 60 days.Values represent the mean and standard deviation (n = 3 independent samples).Adhesive performance Supplementary Fig. 11 Adhesive performance.(a) Lap-shear adhesive performance of CP and CPB adhered to wet porcine tissues from various organs (skin, heart, stomach, liver).(b) Modified 180° peel adhesive performance of CP and CPB adhered to wet porcine tissues from various organs (skin, heart, stomach, liver).